1. Field of Invention
This invention relates to systems and methods, chemical and biological analysis and, in particular, to systems, apparatus, and methods of sample preparation and conditioning involving integrated high hydrostatic pressure treatment with flow-through analytical systems and methods that facilitate separation or extraction or chemical reaction of components of a sample.
2. Discussion of Related Art
Advancements in chemical and biological analysis have been driven by analytical and separation equipment. However, the first step in analytical processes, sample preparation, has received little attention and has predominantly focused on off-line traditional mechanical shearing or chemical approaches at various temperatures. Most analytical instruments require true solutions of the analytes as an input, while most samples, particularly biological and environmental samples, contain cells, tissues, suspensions, emulsions and other heterogeneous compositions. The majority of published methods combine modern state-of-the-art high sensitivity and high resolution analytical methods with the legacy sample preparation steps. Most sample preparation protocols commonly used have been developed before modern molecular analysis methods, such as mass spectrometry, DNA sequencing and PCR amplification techniques, existed. Many sample preparation methods in common use continue to rely on traditional techniques such as mechanical homogenization, ultrasonic cavitational disruption, grinding of frozen samples in liquid nitrogen, etc. Most of these techniques require processing samples one-by-one in a dedicated container, leading to the necessity of manual sample handling or the use of robotic liquid handlers. Sample transfer typically presents a risk of undesired sample loss, potential for operator error, sample cross-contamination, and overall lack of an automated in-line process from initial sample to results.
Thermodynamic control of molecular interactions and chemical equilibria could be accomplished by varying the two orthogonal parameters of temperature and pressure. Temperature has been by far the most widely used perturbation in biochemical thermodynamics. However, a complete thermodynamic response can be utilized by using pressure perturbations, which is governed by different thermodynamic effects than temperature.
Hydrostatic pressure has been used to promote cell lysis, extraction and partitioning of various molecular entities as exemplarily illustrated by Lazarev et al. in U.S. Patent Application Publication No. 2008/0300386 A1, which is incorporated herein by reference in its entirety for all purposes. The control of molecular interactions has also been disclosed as noted by Litt et al. in U.S. Pat. No. 6,635,469 B1, which is also incorporated herein by reference in its entirety for all purposes. Enzymatic reactions, including proteolysis for preanalytical sample preparation in mass spectrometry-based proteomics have also been disclosed by, for example, Laugharn et al. in European Patent Specification No. EP 0 814 900 B1, which is incorporated herein by reference in its entirety for all purposes, and by Lopez-Ferrer in U.S. Patent Application Publication No. 2009/0203068 A1. To date, the application of hydrostatic pressure to liquid samples has been predominantly achieved by pressurizing samples contained in closed pressure vessels. Such techniques may not be practical for pressurization of very small volume liquid samples in the micro liter range and do not interface well with automated analysis systems.
Flow-through high pressure reactor apparatus has been described by Laugharn et al. in U.S. Pat. No. 6,036,923, which is incorporated herein by reference in its entirety for all purposes, which allows loading and unloading operations to be automated by the use of the high-pressure valves to trap the sample in a segment of the tubular flow path, enabling a variety of applications, ranging from chromatography at high pressure to control of enzyme kinetics under pressure. The design of the reactor described above may not accommodate miniaturization and the volumes of samples which could be pressurized has remained relatively large (1 ml and above). The alternative method of pressurization of small samples has also been described by, for example, Lopez-Ferrer in U.S. Patent Application Publication No. 2009/0203068 A1. However, such approach is limited in a way that the sample material is placed in direct contact with the liquid used as a source of hydrostatic pressure through the series of valves, and which poses the risk of sample cross-contamination when processing of samples is conducted in a serial fashion. Furthermore such approaches can only be pressurized to the maximum pressure level available on the LC system and the sample pressure cannot be easily controlled to slowly ramp or rapidly cycle pressure as a function of time.